Bacterial genomic DNA in 1%-agarose-Gel? - ... is it genomic DNA oder just plasmid-DNA (Mar/21/2006 )
I extracted DNA from bacteria, made a specific PCR-amplification - everything worked fine!
I my agarose-gel (of the extract) there is a band that i supposed to be genomic DNA - today i calculated the size of the DNA in this band (with help of a half-logarithmic plot), and came to the result that it is just 15-19 kbp! - A bit small, isn't it? (As referance i had a ladder from 0.1 to 2 kbp - i used a 1%agarose-Gel - 45minutes at 120V and 500mA)
I asked the people in the lab where i currently work, and they told me, that this is genomic and not plasmid-DNA. They told me that the reason for this are conformational effects, that make the DNA compact and run faster,...
For me its hard to belive, because the difference between the size i expect and the size i calculated is the factor 10² (=100).
Whats your opinion,...?
IMO, that standard (that only goes up to 2kbp) is no help at all in determining the Mw of your gDNA
think about the size of a bacterial chromosome; think about conformational changes, think about the scale you are dealing with...I would not expect to get a crisp, accurately-weighted band when running gDNA
I can tell you from experience that if your gDNA is pretty good, you will see a whopping band up by the top of the gel (btw, 1% is pretty high for a piece of DNA that large) no matter what sort of standard you use and how you run it. there should be minimal smearing if it's a good prep and you didn't shear your sample. you can see an example at this link of what you should see (note that a couple are a little smeared; you want the ones with a bit more resolution)
hope this helps
What gel-conc did you use to detect your gDNA?
What kind of ladder did you use?
that pic I sent was a link to a website
if I want to check gDNA, I usually run a 0.5% gel, not too high voltage. the standard doesn't matter so much, just should be larger (I use lambda/hindIII routinely; the largest band is around 23kb)
but this is not to get a MW; like I said you can't really be accurate for that purpose. I check for a fairly discrete band that is very large on the gel, with minimal/no smearing...this tells me the prep is good. it is not for getting a MW